Near‐infrared‐induced drug release from antibody–drug double conjugates exerts a cytotoxic photo‐bystander effect

Abstract Ideal cancer treatments specifically target and eradicate tumor cells without affecting healthy cells. Therefore, antibody‐based therapies that specifically target cancer antigens can be considered ideal cancer therapies. Antibodies linked with small‐molecule drugs (i.e., antibody–drug conjugates [ADCs]) are widely used in clinics as antibody‐based therapeutics. However, because tumors express antigens heterogeneously, greater target specificity and stable binding of noncleavable linkers in ADCs limit their antitumor effects. To overcome this problem, strategies, including decreasing the binding strength, conjugating more drugs, and targeting tumor stroma, have been applied, albeit with limited success. Thus, further technological advancements are required to remotely control the ADCs. Here, we described a drug that is photo‐releasable from an ADC created via simple double conjugation and its antitumor effects both on target and nontarget tumor cells. Specifically, noncleavable T‐DM1 was conjugated with IR700DX to produce T‐DM1‐IR700. Although T‐DM1‐IR700 itself is noncleavable, with NIR‐light irradiation, it can release DM1‐derivatives which elicited antitumor effect in vitro mixed culture and in vivo mixed tumor model which are mimicking heterogeneous tumor‐antigen expression same as real clinical tumors. This cytotoxic photo‐bystander effect occurred in various types mixed cultures in vitro, and changing antibodies also exerted photo‐bystander effects, suggesting that this technology can be used for targeting various specific cancer antigens. These findings can potentially aid the development of strategies to address challenges associated with tumor expression of heterogeneous antigen.

Japan Society for the Promotion of Science, Grant/Award Numbers: 18K15923, 21K07217

| INTRODUCTION
Controlled and localized targeted drug delivery is ideal for minimizing systemic toxicity and inducing highly localized therapeutic effects. 1 Because antibody-drug conjugates (ADCs) can selectively deliver drugs to cells expressing a target antigen, they are considered an ideal modality for treating various diseases, especially cancer. [2][3][4] However, ADCs can get degraded before reaching the target site due to linker instability, resulting in reduced efficacy against the targets and increased systemic toxicity. [4][5][6] Moreover, the pharmacokinetics and biodistribution of ADCs can be affected if multiple drugs are conjugated to the antibody, resulting in ADC degradation in the liver. 7 Therefore, it is important to use appropriate linkers and drugto-antibody ratios (DARs). The linkers between antibodies and drugs in clinically administered ADCs remain stable in the plasma when these ADCs have DARs of three or four. 2,8 Furthermore, stable linkers enhance the specificity of antibodies, thereby reducing the systemic toxicity of ADCs, which is called as a noncleavable linker. Although ADCs have shown success in treating blood cancers, their success has been limited against solid tumors. Unlike hematological malignancies, solid tumors are difficult to target, because the target antigens are heterogeneously expressed by tumor cells. 9,10 Thus, further technological advancements are required to develop remotely controlled ADCs that accumulate in targeted tumor lesions and widely release the drugs therein to eradicate both target and nontarget tumor cells. [11][12][13] Trastuzumab (Tra) emtansine (T-DM1) is a clinically applied ADC containing Trastuzumab (Tra), an HER2-targeting humanized monoclonal antibody (mAb). 14 Tra is covalently linked to the cytotoxic agent DM1 (a maytansinoid, cytotoxic component that binds to the ends of microtubules) with an N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker (a thioether-linked, noncleavable linker). T-DM1 is catabolized in lysosomes after receptormediated internalization by HER2-expressing cancer cells, resulting in the release of DM1-containing catabolites that subsequently bind to tubulin and cause mitotic arrest and apoptosis. 4 Due to the noncleavable linker (SMCC) with thiol band in T-DM1, this ADC is cytotoxic only after cellular internalization, and thus, does not exert bystander cytotoxic effects on nontarget cells. 3,15,16 Therefore, T-DM1 cannot fully eradicate solid tumors due to heterogeneous HER2 expression. 3,16 Near-infrared (NIR)-photoimmunotherapy (PIT) is a recently developed cancer therapy that involves exposing an antibodyphotoabsorber conjugate to NIR light. 17 An antibody-photoabsorber conjugate comprises a cancer cell-specific mAb that is covalently conjugated to IRDye 700DX NHS ester (IR700), a silica-phthalocyaninederived photoabsorber that binds the cell-surface target antigen and induces necrosis after exposure to 690 nm NIR light. [18][19][20][21] This novel therapy is currently undergoing an international phase III clinical trial and was recently conditionally approved by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan.
Recent research revealed that the mechanism of NIR-PIT was rapid necrotic cell death due to photochemical ligand reactions of IR700. [22][23][24] This photochemical reaction changes the hydrophilic side chains (silanol) of IR700 into hydrophobic, which introduces the aggregation of the antibody-IR700 conjugates. With this unique mechanism for specific cell membrane ruptures, NIR-PIT is thought to be a new modality in cancer therapy. However, since NIR-PIT exploited the antibody targeting ability, its highly selective cytotoxicity on the target-expressing cells limits on the whole antitumor effect on heterogeneous antigen-expressing tumors. 25,26 In this study, we created a simple double-conjugated ADC comprising T-DM1 and IR700 to enable remotely controlled drug release from ADCs ( These results effectively demonstrated the concept of a "cytotoxic photo-bystander effect" in mixed tumors ( Figure 1).

| Production of T-DM1-IR700 and Tra-IR700
In T-DM1, Tra is conjugated to the maytansinoid DM1-a cytotoxic tubulin inhibitor-, 27 which comprises an SMCC linker (noncleavable) and mertansine (a thiol-containing maytansinoid) ( Figure 1). These structures facilitate the endocytosis of T-MD1 after HER2 binding, followed by its degradation and the intracellular release of DM1. 4,14 In this study, we conjugated T-DM1 to IR700 to generate the simple doubleconjugate T-DM1-IR700 (Figure 2a), whose release was then evaluated. Trastuzumab-IR700 (Tra-IR700) was also produced as a control with the conjugation of trastuzumab and IR700. The successful conjugation of Tra and T-DM1 to IR700 was confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IR700-based fluorescence imaging (FLI) (Figure 2a). The number of IR700-dye to mAb molecules was adjusted so that approximately three IR700 molecules were conjugated on per one mAb molecule.
F I G U R E 1 Legend on next page.

| NIR-light triggered drug releasing from T-DM1-IR700
We first characterized the NIR-light triggered drug releasing from T-DM1-IR700 (Figure 2b). Cytotoxicity on NIR-PIT is based on a photochemical reaction involving silanol ligand release, which makes hydrophilic conjugates hydrophobic and results in their aggregation. 22,28 Previous data demonstrated that silicon phthalocyanines undergo axial ligand exchange especially in hypoxic conditions and generate toxic reactive oxygen species (ROS) especially in the normoxic conditions. 29,30 SDS-PAGE revealed the formation of nonfluorescent, irradiated T-DM1-IR700 aggregates with sizes larger than those of IgG; however, IR700-fluorescence was dose-dependently quenched by NIRlight ( Figure 2c). Thus, after NIR irradiation, T-DM1-IR700 became aggregated and lost IR700-fluorescence.
We then analyzed the photo-release of DM1 derivatives from the T-DM1-IR700 conjugates in tube (Figure 2d, left panel). Since the T-DM1 is based on noncleavable thiol linker, no specific peak was detected from the supernatants without NIR-light ( Figure 2d). After NIR-light irradiation (16 J/cm 2 ), a specific peak was detected by highperformance liquid chromatography (Figure 2d), and the relative volume of photo-released substrates was dose-dependently elevated by NIR-light, and a plateau was observed at 8 J/cm 2 (Figure 2e).
We then compared the substrates from the irradiated sample with S-ME-DM1 using mass spectroscopy (MS) and identified the substrates as DM1 derivatives by product ion analysis using a highresolution mass spectrometer with further fragmentation analysis (Figures 2f and S1). 31 Additionally, we performed LC-MS/MS with aMFc-DM1-IR700 (with a different antibody) and successfully detected photo-released substrates. Each peak has similar retention time, suggesting that they were DM1 derivatives ( Figure S2). Collectively, these results identified the DM1 derivatives were photoreleased from T-DM1-IR700. Specifically, the photo-release reaction was universal, irrespective of the changes in the antibody.
To determine whether the releasing the DM1 derivatives depended on NIR-light triggered silanol release from IR700, 29,30 or ROS generation from IR700 with NIR-light, we added an ROS quencher (sodium azide [NaN 3 ]) or an electron donor (L-cysteine) to the tube and evaluate the relative volume of photo-released substrates ( Figure 2g). We found that the relative volume of the photoreleased DM1 derivatives was partially decreased in the presence of NaN 3 and almost fully blocked with L-cysteine, suggesting that ROS generation was primarily involved in DM1 derivatives' photo-release with cleavage of the linker.

|
In vitro assessment of NIR-PIT with Tra-IR700 and T-DM1-IR700, and "cytotoxic photobystander effect" from the released DM1 derivatives on in vitro mixed cell culture Next, Tra-IR700 and T-DM1-IR700 bound to HER2-expressing 3T3/ HER2 cells (HER2+), whereas these binding events were blocked by excess Tra or T-DM1, respectively, indicating that they specifically  Figure S4). Moreover, Tra-IR700 inhibited the F I G U R E 1 Scheme of NIR-triggered drug release exerting a cytotoxic photo-bystander effect on a mixed tumor (mimicking a tumor that expresses heterogeneous target antigens) and Schematic representation of mAb-DM1-IR700 (double-conjugated antibody). Schematic of the cytotoxic photo-bystander effect of photo-triggered drug release and an ADC conjugated to IR700. First, the dual conjugate (the ADC conjugated to IR700) was prepared for intravenous injection. The agents then accumulated near the targeted tumor antigen inside the tumor, which heterogeneously expressed the targeted antigens. Upon NIR-light exposure, the targeted tumor cells were ruptured via NIR-PIT (photo-necrosis). At the same time, the conjugated drugs were released around the ruptured cells, and the drugs released in response to the photochemical reaction scattered to nontargeted tumor cells and induced cytotoxicity in the remaining live tumor cells. Schematic representation of mAb-DM1-IR700. DM1 was linked to any mAb with the noncleavable thiol based SMCC linker. mAb-DM1 was then double-conjugated with IR700 to generate mAb-DM1-IR700. We then evaluated the effects of NIR-PIT (4 J/cm 2 ) with Tra-IR700 or T-DM1-IR700 on an in vitro mixed culture of HER2+ F I G U R E 2 Legend on next page. 3T3/HER2 and HER2À MDAMB-468-luc-GFP cells (Figure 3d). Immediately after NIR-light irradiation, neither Tra-IR700-nor T-DM1-IR700-mediated NIR-PIT exerted an effect on nontargeted MDAMB-468-luc-GFP cells in the mixed culture ( Figure S5). This specific photocytotoxicity was consistent with that described previously. 18,32 MDAMB-468-luc-GFP cells treated with T-DM1-IR700 did not show any significant decrease in luciferase activity at 4 days after irradiation ( Figure S6). No in vitro significant effects on nontargeting cells (MDAMB-469-luc-GFP) in mixed culture was detected with T-DM1 incubation, and a mixture of Tra-IR700 and T-DM1 with NIR-light grew nontargeting cells more than that without NIR-light or control.
NIR-PIT with Tra-IR700 and T-DM1 (NIR-PIT) eradicated 3 T3/HER2 cells from the mixed culture, resulting in diminishing contact inhibition (making more space), which grew nontargeting cells (MDAMB-468-luc-GFP) more ( Figure S7). Four days after NIR-PIT, Tra-IR700-mediated NIR-PIT eradicated most of the 3T3/HER2 cells, resulting in making more new space, thereby allowing the MDAMB-468-luc-GFP cells to grow more than that observed under control conditions. Moreover, Tra-IR700 alone inhibited the growth of 3T3/HER2 cells to facilitate more space to grow MDAMB-468-luc-GFP cells than that observed with the controls. Furthermore, the relative light unit (RLU) ratio of Tra-IR700-mediated NIR-PIT was greater than that of Tra-IR700 alone, suggesting that NIR-PIT almost killed Therefore, we made mixed tumor model mimicking this heterogeneous expression of HER2 in human lung cancer resected specimens.  Figure S1. (g) MS data obtained following inhibition of the specific peak (DM1) in an NIR-light-irradiated (16 J/cm 2 ) tube. The relative ratio (defined 0 J/cm 2 tube = 1) is shown (n = 3). The data represent the mean ± standard deviation. *p < 0.05 (Kruskal-Wallis test with Dunn's post hoc test) specific localization of IR700 fluorescence in tissues other than liver and urinary bladder, presumably owing to hepatic metabolism and urinary excretion. The Tra-IR700: T-DM1-IR700 fluorescence ratios did not differ significantly at any time point, suggesting that their biodistributions were almost entirely dependent on Tra and that the effects of the conjugated drugs were negligible (Figure 4c). Ex vivo F I G U R E 3 Legend on next page. biodistribution study revealed that lung and heart had some IR700-fluorescence until 12 h compared to tumors or livers. The Tra-IR700 or T-DM1-IR700 fluorescence on ex vivo tumors did not differ significantly at any time point ( Figure S10). The analysis of frozen tumor section revealed that Tra-IR700 or T-DM1-IR700 specifically accumulated on the HER2 expressing tumor regions (non-GFP regions) in mixed ex vivo tumors ( Figure S11). These data indicated that Tra-IR700 and T-DM1-IR700 were specifically accumulated in mixed tumors, and that NIR-light irradiation 1-2 days postadministration might be appropriate. Moreover, IR700 fluorescence could be used as a marker to indicate the site of NIR-light irradiation.

| Cytotoxic photo-bystander effect on in vivo
We then tested the in vivo cytotoxic photo-bystander effects on mixed tumors (Figure 4d  Preincubation with excess Tra or T-DM1 inhibited the binding of Tra-IR700 or T-DM1-IR700 to 3T3/HER2 cells, respectively, indicating that Tra-IR700 and T-DM1-IR700 specifically bound to the HER2 antigen. Neither Tra-IR700 nor T-DM1-IR700 showed IR700 fluorescence signals in the presence of HER2ÀMDAMB-468 cells. (b) Microscopic observations before and immediately after HER2-targeted NIR-PIT. Mixed cultures of HER2+ 3T3/HER2 and HER2À MDAMB-468-luc-GFP cells were incubated with Tra-IR700 or T-DM1-IR700 overnight and observed under a microscope before and immediately after irradiation with NIR light (4 J/cm 2 ). Necrotic cell death (revealed using PI staining) was observed only for HER2+ 3T3/HER2 cells after NIR light exposure, whereas HER2À MDAMB-468-luc-GFP cells remained intact. Confirmation of the selective cytotoxicity induced by NIR-PIT with both Tra-IR700 and T-DM1-IR700. Scale bars: 20 μm. (c) In vitro NIR-PIT (4 J/cm 2 ) with Tra-IR700 (1 μg/ml) or T-DM1-IR700 (1 μg/ml) on HER2+ (3T3/HER2-luc-GFP) and HER2À (MDAMB-468-luc-GFP) cells. Luciferase activities were measured as RLU values (n = 4, *p < 0.001). (d) Co-culture of 3T3/HER2 and MDAMB-468-luc-GFP cells. NIR-PIT was performed following treatment with T-DM1-IR700 (1, 5, or 10 μg/ml) or Tra-IR700 (10 μg/ml), after which the mixed cultures were incubated for 4 days (left panel). Luciferase activities were measured as RLUs, and the viability of nontargeted MDAMB-468-luc-GFP cells was measured 4 days after NIR-light irradiation. Upon NIR-PIT following Tra-IR700 treatment of the mixed culture, HER2+ 3T3/HER2 cells were eradicated, resulting in more space available for the nontargeted MDAMB-468-luc-GFP cells to grow (n = 4). NIR-PIT showed no immediate effect on MDAMB-468-luc-GFP cells in the mixed culture ( Figure S5). Only MDAMB-468-luc-GFP cells treated with T-DM1-IR700 did not show any significant decrease in luciferase activity 4 days after irradiation ( Figure S6). Other cell-line combinations (HER2+ and HER2À or -low cells) were also examined and showed cytotoxic photo-bystander effects ( Figure S8). In panels (e) and (f), the data are presented as the mean ± standard error of the mean (SEM). In panel (g), the data are presented as the mean ± standard deviation. *p < 0.0001, **p < 0.01 (Student's t test).   (Figure 1). This photo-bystander cytotoxic effect is unspecific; therefore, the photo-released cytotoxic drugs could give damage on adjacent normal cells. Moreover, in the tumor microenvironment, anti-tumor immune cells have an important role for the effective immunotherapy. Unspecific photo-bystander effect might affect these immune cells, which might be a concern for this technology of photo-triggered drug releasing. However, the NIR-light irradiation is done after the ADCs are specifically accumulated on targeted tumor site, the damage on normal cells could be controlled to be minimized.
We successfully developed an in vivo photo-released drug system involving the use of NIR light. Drug release was spatiotemporally regulated using NIR light, which can penetrate tissues to a deeper extent than light of other wavelengths, without damaging the normal tissues.
Additionally, we used ADCs to target tumor antigens and achieve accumulation in tumor lesions, resulting in the release of drugs at high concentrations upon NIR-light irradiation of the tumor area. These findings indicated that drugs with high toxicity can be efficiently utilized. Interestingly, this method employs extremely simple chemistry for conjugating IR700 to ADCs, and we further demonstrated the effectiveness of the method using different antibodies; therefore, we could exploit this drug releasing technology for a variety of cancers.
Notably, targeting molecules (peptides and/or ligands) can also be used in this approach. Furthermore, this method is easily translatable to the clinic, given that T-DM1 has already been clinically approved, 15,33 and NIR-PIT was recently approved by the PMDA in Japan. Finally, this method can be applied to fields other than those related to cancer therapy.

| CONCLUSION
In conclusion, we demonstrated a system involving NIR-light-triggered drug release in a heterogeneous tumor for efficient eradication of tumor cells. The accumulation of IR700 fluorescence of the photoabsorber in conjugates could also be used as a noninvasive guide for determining the therapeutic area and as a biomarker to confirm the efficacy of therapy. Furthermore, as this system enables target switching by changing the mAbs or drugs, it can be used as a potential novel platform for photo-controlled drug release. This technology can also be readily translated to clinical settings based on the recent approval of NIR-PIT for therapeutic applications in 2020 and the current use of T-DM1 in clinical practice.    The concentration of IR700 was calculated based on the absorption at 689 nm to confirm the number of IR700 molecules conjugated to the antibody. 36,39 The number of mAb molecules was adjusted to approximately three IR700 molecules per one mAb molecule.

| Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDS-PAGE was performed using 4%-20% Tris-Glycine mini gels (Thermo Fisher Scientific) to confirm mAb-IR700 conjugation. 40,41 The fluorescent bands were visualized with a Pearl Imager (LI-COR Bioscience), using the 700 nm fluorescence channel after electrophoresis for 90 min at 20 mA. The gel was stained with Colloidal Blue (Thermo Fisher Scientific) to confirm the molecular weight of the mAb-IR700 conjugates.

| Immunostaining
HER2 immunostaining was performed on surgically resected specimens derived from patients who underwent surgery at Nagoya University Hospital from April 2011 to December 2015 and who were diagnosed with lung adenocarcinoma, squamous cell carcinoma, or SCLC by pathologists. For the in vivo experiments, mixed tumors from mice were harvested, and 4-μm thick formalin-fixed, paraffin-embedded sections were prepared. A Histofine HER2 Kit MONO (Nichirei Biosciences, Inc., Tokyo, Japan) was used to detect the HER2 protein, according to manufacturer instructions. HER2 expression was evaluated according to the guidelines for HER2 staining in breast cancer tissues.
For NIR-PIT, cells were irradiated with 4 J/cm 2 of NIR light from a 690 nm-Laser (MLL-III-690, Changchun New Industries Optoelectronics Tech, CO., Ltd, Changchun, China). The actual power density (mW/cm 2 ) was determined using an optical power meter (PM100; Thorlabs, Newton, NJ, USA), as previously reported. 42 Cell viability was evaluated based on luciferase activity.
For the luciferase assay for in vitro mixed culture, 200 μl of 150 μg/ml D-luciferin-containing media was added to PBS-washed cells.
For evaluating NIR-PIT in vitro on the single cell line (3T3/HER2 or MDAMB468-luc) in Figure 3c, after washing twice with PBS, cells were irradiated with 4 J/cm 2 of NIR light and at 1 h later, cell viability was evaluated.
For evaluating NIR-PIT in vitro on the mixed cell lines (Figure 3d and Figure S8), after washing twice with PBS, replacing phenol red free media 300 μl, then cells were irradiated with 4 J/cm 2 of NIR light.
At 4 days after NIR-PIT, the viability of luciferase-expressing cells was evaluated using a bioluminescence plate reader (Powerscan 4).

| In vivo NIR-PIT
Tra-IR700 (3.6 μg/g) or T-DM1-IR700 (3.6 μg/g) was administered intravenously to mice on Day À1 (6 days after tumor cell transplantation). The dose similar to that of T-DM1 administered to humans (3.6 mg/kg). The NIR-light was irradiated at 1 and 2 days after the drug administration (Figure 4d). 43,44 The tumors on mice were then

| In vivo FLI
Tra-IR700 was administered intravenously to mice, and the IR700 FLI was assessed using a Pearl Imager (LI-COR Biosciences). 45

| Preparation for the in vitro MS samples
For detecting released DM1 derivatives in tube, the T-DM1-IR700 (500 μg/ml) in PBS was irradiated with a 690 nm NIR laser. The sample was centrifuged with 10 K membrane Amicon Ultra (Merk, Darmstadt, Germany) to remove mAb-derived proteins, and the 10 K passed solution was analyzed with the MS.
For detecting released DM1 derivatives in vitro mixed culture, the 10 μg/ml T-DM1-IR700 in PBS was incubated with the mixed cells for 6 h. After washing cells twice with PBS, cells were NIR-light irradiated.
The supernatant of the mixed cell culture was collected, then the sample was centrifuged with 10 K membrane Amicon Ultra to remove proteins, and the 10 K passed solution was analyzed with the MS.

| Quantification and statistical analysis; statistical analysis
Data are expressed as the mean ± SEM of a minimum of three experiments, unless otherwise indicated. Statistical analyses were conducted using Prism software (GraphPad Software, San Diego, CA, USA). For two-group comparisons, a Student's unpaired t-test was used. For multiple-group comparisons, one-way analysis of variance with Tukey's test or Dunnett's test was used. The cumulative probability of survival, defined as the nonachievement of a tumor diameter of 20 mm, was estimated in each group using Kaplan-Meier analysis, and the results were compared using the log-rank and Wilcoxon tests. p < 0.05 was considered to reflect a statistically significant difference.